West Nile Virus (WNV) has recently emerged as a major US public health concern due to the outbreak of human encephalitis in the USA in 2002 with reported 4165 WNV cases and 284 deaths. Additionally, recent findings of virus transmission by blood transfusion, organ transplantation, and intrauterine infection indicate a need for WNV testing of blood donor specimens. Therefore, the FDA has recommended to screen any blood supply for WNV. Tests which are specific and highly sensitive and which can identify an acute infection with ongoing viremia are in need for such a screening process.
WNV is taxonomically classified within the family Flaviridae, genus Flavivirus. For WNV two genetic lineages have been described, lineage 1 having world-wide distribution, ranging from West Africa to the Middle East, Eastern Europe, North America, and Australia. Lineage 2 consists exclusively of strains from Africa which have been isolated only in sub-Saharan Africa and Madagascar.
WNV is arthropod-borne and mainly affects birds in their natural reservoir. Some species, such as the American crow, seem particularly susceptible. Susceptible mammalian species, including horses, dogs, and humans, are incidentally infected through insect bites.
The genome of WNV is a single-stranded plus-sense RNA of approximately 11000 nucleotides. It consists of a 5′ non-coding region (NCR, approximately 100 nucleotides), a single open reading frame coding for three viral structural proteins (capsid or core (C), premembrane (prM) and membrane (M), envelope (E)), seven non-structural proteins, and a 3′ NCR (approximately 600 nucleotides). The RNA lies within an internal capsid, which is composed of multiple copies of the core protein and is surrounded by an outer, host derived lipid membrane containing the viral envelope and membrane structural proteins which are responsible for many important properties of the virus, including host range, tissue tropism, replication, assembly, and stimulation of B and T cell immune responses. The seven non-structural proteins are involved in viral replication, maturation, and packaging.
WNV is a member of the Japanese encephalitis virus group, which contains Japanese encephalitis (JE), St. Louis encephalitis (SLE), Murray Valley encephalitis (MVE) and Kunjin virus (an Australian subtype of WNV). The close antigenic relationship of the flaviviruses, particular those belonging to the Japanese encephalitis complex, accounts for the serologic cross-reaction observed in the diagnostic laboratory.
WNV diagnostic testing is often based on the detection of immunoglobulin M (IgM) antibodies to WNV. In at least 90% of the infected patients, IgM antibodies against WNV can be detected in sera or cerebral spinal fluid collected on or 8 days after the onset of the disease using an IgM capture Enzyme-Linked Immunosorbent Assay (ELISA). Once developed, IgM antibodies persist for more than 6 months after disease in over 50% of the patients. Due to the persistence of IgM antibodies to WNV, a positive test for IgM is not necessarily a result of an acute infection by WNV. WNV antibodies are known to cross-react with other flaviviruses, which can make the unequivocal identification of WNV difficult. Plaque reduction neutralization assays can be performed to help distinguish among the flaviviruses. Other tests which target the WNV genome and are therefore highly specific are those comprising the enzymatic amplification of nucleic acids. These tests can be used to document minute amounts of virus in blood or tissues of an individuum.
Lanciotti et al. (J. Clin. Microbiol. (2000) 38, 4066-4071) describe the detection of WNV RNA in host organisms by a RT-PCR assay using nucleotide sequences which bind to sequences encoding the core and the pre-membrane proteins of WNV, by a TaqMan assay using nucleotide sequences which bind to the 3′ non-coding region of WNV, and by a TaqMan assay using nucleotide sequences which bind to the sequence encoding the envelope protein of WNV. Lanciotti and Kerst (J. Clin. Microbiol. (2001) 39, 4506-4513) describe the detection of WNV RNA by a nucleic acid sequence based amplification (NASBA) assay using nucleotide sequences which bind to sequences encoding the envelope protein of WNV. Shi et al. (J. Clin. Microbiol. (2001) 39, 1264-1271) describe the detection of WNV RNA by a real-time RT-PCR assay using nucleotide sequences which bind to sequences encoding the envelope protein, the non-structural protein 1, and the 3′ non-coding region of WNV. Porter et al. (Am. J. Trop. Med. Hyg. (1993) 48, 440-446) describe the detection of WNV RNA by a RT-PCR assay using nucleotide sequences which bind to sequences encoding the non-structural protein 3 of WNV. Briese et al. (Lancet 355 (2000),1614-1615) describe the detection of WNV RNA by a TaqMan assay using nucleotide sequences which bind to sequences encoding the non-structural protein 3 and the non-structural protein 5 of WNV. Hadfield et al. (Mol. Cell Probes 15 (2001), 147-150) describe the detection of WNV RNA by a TaqMan assay using nucleotide sequences which bind to sequences encoding the non-structural protein 3 of WNV.